Thermophilic bacteria are organisms which are capable of growth at elevated temperatures. Unlike the mesophiles, which grow best at temperatures in the range of 25–40° C., or psychrophiles, which grow best at temperatures in the range of 15–20° C., thermophiles grow best at temperatures greater than 50° C. Indeed, some thermophiles grow best at 65–75° C., and some of the hyperthermophiles grow at temperatures up to 130° C. (e.g., J. G. Black, Microbiology Principles and Applications, 2d edition, Prentice Hall, New Jersey, 1993, p. 145–146).
The thermophilic bacteria encompass a wide variety of genera and species. There are thermophilic representatives included within the phototrophic bacteria (i.e., the purple bacteria, green bacteria, and cyanobacteria), eubacteria (i.e., Bacillus, Clostridium, Thiobacillus, Desulfotomaculum, Thermus, lactic acid bacteria, actinomycetes, spirochetes, and numerous other genera), and the archaebacteria (i.e., Pyrococcus, Thermococcus, Thermoplasma, Thermotoga, Sulfolobus, and the methanogens). There are aerobic, as well as anaerobic thermophilic organisms. Thus, the environments in which thermophiles may be isolated vary greatly, although all of these organisms are isolated from areas associated with high temperatures. Natural geothermal habitats have a worldwide distribution and are primarily associated with tectonically active zones where major movements of the earth's crust occur. Thermophilic bacteria have been isolated from all of the various geothermal habitats, including boiling springs with neutral pH ranges, sulfur-rich acidic springs, and deep-sea vents. In general, the organisms are optimally adapted to the temperatures at which they are living in these geothermal habitats (T. D. Brock, “Introduction: An overview of the thermophiles,” in T. D. Brock (ed.), Thermophiles: General, Molecular and Applied Microbiology, John Wiley & Sons, New York, 1986, pp. 1–16). Basic, as well as applied research on thermophiles has provided some insight into the physiology of these organisms, as well as use of these organisms in industry and biotechnology.
I. Uses For Thermophilic Enzymes
Advances in molecular biology and industrial processes have led to increased interest in thermophilic organisms. Of particular interest has been the development of thermophilic enzymes for use in industries such as the detergent, flavor-enhancing, and starch industries. Indeed, the cost savings associated with longer storage stability and higher activity at higher temperatures of thermophilic enzymes, as compared to mesophilic enzymes, provide good reason to select and develop thermophilic enzymes for industrial and biotechnology applications. Thus, there has been much research conducted to characterize enzymes from thermophilic organisms. However, some thermophilic enzymes have less activity than their mesophilic counterparts under similar conditions at the elevated temperatures used in industry (typically temperatures in the range of 50–100° C.) (T. K. Ng and William R. Kenealy, “Industrial Applications of Thermostable Enzymes,” in T. D. Brock (ed.), Thermophiles: General, Molecular, and Applied Microbiology, 1986, John Wiley & Sons, New York, pp. 197–215). Thus, the choice of a thermostable enzyme over a mesophilic one may not be as beneficial as originally assumed. However, much research remains to be done to characterize and compare thermophilic enzymes of importance (e.g., polymerases, ligases, kinases, topoisomerases, restriction endonucleases, etc.) in areas such as molecular biology.
II. Thermophilic DNA Polymerases
Extensive research has been conducted on isolation of DNA polymerases from mesophilic organisms such as E. coli. (e.g., Bessman et al., J. Biol. Chem. 223:171, 1957; Buttin and Kornberg, J. Biol. Chem. 241:5419, 1966; and Joyce and Steitz, Trends Biochem. Sci., 12:288–292, 1987). Other mesophilic polymerases have also been studied, such as those of Bacillus licheniformis (Stenesh and McGowan, Biochim. Biophys. Acta 475:32–44, 1977; Stenesh and Roe, Biochim. Biophys. Acta 272:156–166, 1972); Bacillus subtilis (Low et al., J. Biol. Chem., 251:1311, 1976; and Ott et al., J. Bacteriol., 165:951, 1986); Salmonella typhimurium (Harwood et al., J. Biol. Chem., 245:5614, 1970; Hamilton and Grossman, Biochem., 13:1885, 1974); Streptococcus pneumoniae (Lopez et al., J. Biol. Chem., 264:4255, 1989); and Micrococcus luteus (Engler and Bessman, Cold Spring Harbor Symp., 43:929, 1979), to name but a few.
Somewhat less investigation has been performed on the isolation and purification of DNA polymerases from thermophilic organisms. However, native (i.e., non-recombinant) and/or recombinant thermostable DNA polymerases have been purified from various organisms, as shown in Table 1 below.
TABLE 1Polymerase Isolation From Thermophilic OrganismsOrganismCitationThermus aquaticusKaledin et al., Biochem., 45:494–501(1980); Biokhimiya 45:644–651 (1980).Chien et al., J. Bacteriol., 127:1550 (1976).University of Cincinnati Master's thesis byA. Chien, “Purification andCharacterization of DNA Polymerase fromThermus aquaticus,” (1976).University of Cincinnati Master's thesisby D. B. Edgar, “DNA Polymerase From anExtreme Thermophile: Thermus aquaticus,”(1974).U.S. Pat. No. 4,889,818*U.S. Pat. No. 5,352,600*U.S. Pat. No. 5,079,352*European Patent Pub. No. 258,017*PCT Pub. No. WO 94/26766*PCT Pub. No. WO 92/06188*PCT Pub. No. WO 89/06691*Thermatoga maritimaPCT Pub. No. WO 92/03556*Thermatoga neapolitanaU.S. Pat. No. 5,912,155*U.S. Pat. No. 5,939,301*U.S. Pat. No. 6,001,645*Thermatoga strainSimpson et al., Biochem. Cell Biol.,FjSS3-B.168:1292–1296 (1990).Thermosipho africanusPCT Pub. No. 92/06200*U.S. Pat. No. 5,968,799*Thermus thermophilusMyers and Gelfand, Biochem., 30:7661(1991).PCT Pub. No. WO 91/09950*PCT Pub. No. WO 91/09944*Bechtereva et al., Nucleic Acids Res.,17:10507 (1989).Glukhov et al., Mol. Cell. Probes4:435–443 (1990).Carballeira et al., BioTech.,9:276–281 (1990).Ruttiman et al., Eur. J. Biochem.,149:41–46 (1985).Oshima et al., J. Biochem.,75:179–183 (1974).Sakaguchi and Yajima, Fed. Proc.,33:1492 (1974) (abstract).Thermus flavusKaledin et al., Biochem.,46:1247–1254 (1981); Biokhimiya 46:1576–1584 (1981).PCT Pub. No. WO 94/26766*Thermus ruberKaledin et al., Biochem.,47:1515–1521 (1982);Biokhimiya 47:1785–1791 (1982).Thermoplasma acidophilumHamal et al., Eur. J. Biochem.,190:517–521 (1990).Forterre et al., Can. J. Microbiol.,35:228–233 (1989).Sulfolobus acidocaladariusSalhi et al., J. Mol. Biol.,209:635–641 (1989).Salhi et al., Biochem. Biophys. Res. Comm.,167:1341–1347 (1990).Rella et al., Ital. J. Biochem.,39:83–99 (1990).Forterre et al., Can. J. Microbiol.,35:228–233 (1989).Rossi et al., System. Appl. Microbiol.,7:337–341 (1986).Klimczak et al., Nucleic Acids Res.,13:5269–5282 (1985).Elie et al., Biochim. Biophys. Acta951:261–267 (1988).Bacillus caldotenaxJ. Biochem.,113:401–410 (1993).Bacillus stearothermophilusSelimann et al., J. Bacteriol.,174:4350–4355 (1992).Stenesh and McGowan, Biochim.Biophys. Acta 475:32–44 (1977).Stenesh and Roe, Biochim.Biophys. Acta 272:156–166 (1972).Kaboev et al., J. Bacteriol.,145:21–26 (1981).MethanobacteriumKlimczak et al., Biochem.,thermoautotropicum25:4850–4855 (1986).Thermococcus litoralisKong et al., J. Biol. Chem. 268:1965 (1993)U.S. Pat. No. 5,210,036*U.S. Pat. No. 5,322,785*Anaerocellum thermophilusAnkenbauer et al., WO 98/14588*Pyrococcus sp. KOD1U.S. Pat. No. 6,008,025*Pyrococcus furiosusLundberg et al., Gene 108:1 (1991)PCT Pub. WO 92/09689U.S. Pat. No. 5,948,663U.S. Pat. No. 5,866,395*Herein incorporated by reference.
In addition to native forms, modified forms of thermostable DNA polymerases having reduced or absent 5′ to 3′ exonuclease activity have been expressed and purified from T. aquaticus, T. maritima. Thermus species sps17, Thermus species Z05, T. thermophilus, Bacillus stearothermophilus (U.S. Pat. Nos. 5,747,298, 5,834,253, 5,874,282, and 5,830,714) and T. africanus (WO 92/06200).
III. Uses For Thermophilic DNA Polymerases
One application for thermostable DNA polymerases is the polymerase chain reaction (PCR). The PCR process is described in U.S. Pat. Nos. 4,683,195 and 4,683,202, the disclosures of which are incorporated herein by reference. Primers, template, nucleoside triphosphates, appropriate buffer and reaction conditions, and polymerase are used in the PCR process, which involves multiple cycles of denaturation of target DNA, hybridization of primers to the target DNA and synthesis of complementary strands. The extension product of each primer becomes a template in the subsequent cycle for production of the desired nucleic acid sequence. Use of a thermostable DNA polymerase enzyme in PCR allows repetitive heating/cooling cycles without the requirement of fresh DNA polymerase enzyme at each cooling step because heat will not destroy the polymerase activity. This represents a major advantage over the use of mesophilic DNA polymerase enzymes such as Klenow in PCR, as fresh mesophilic polymerase must be added to each individual reaction tube at every cycle. The use of Taq in PCR is described in U.S. Pat. No. 4,965,188, EP Publ. No. 258,017, and PCT Publ. No. 89/06691, herein incorporated by reference.
In addition to PCR, thermostable DNA polymerases are widely used in other molecular biology techniques including recombinant DNA methods. For example, various forms of Taq have been used in a combination method which utilizes reverse transcription and PCR (e.g., U.S. Pat. No. 5,322,770, herein incorporated by reference). DNA sequencing methods utilizing Taq DNA polymerase have also been described (e.g., U.S. Pat. No. 5,075,216, herein incorporated by reference).
However, some thermostable DNA polymerases have certain characteristics (e.g., 5′ to 3′ exonuclease activity) which are undesirable in PCR and other applications. In some cases, when thermostable DNA polymerases that have 5′ to 3′ exonuclease activity (e.g., Taq, Tma, Tsps17, TZ05, Tth and Taf) are used in the PCR process and other methods, a variety of undesirable results have been observed, including a limitation of the amount of PCR product produced, an impaired ability to generate long PCR products or to amplify regions containing significant secondary structure, the production of shadow bands or the attenuation in signal strength of desired termination bands during DNA sequencing, the degradation of the 5′ end of oligonucleotide primers in the context of double-stranded primer-template complex, nick-translation synthesis during oligonucleotide-directed mutagenesis and the degradation of the RNA component of RNA:DNA hybrids. When utilized in a PCR process with double-stranded primer-template complex, the 5′ to 3′ exonuclease activity of a DNA polymerase may result in degradation of oligonucleotide primers from their 5′ end. This activity is undesirable not only in PCR, but also in second-strand cDNA synthesis and sequencing processes.
When choosing to produce and use an enzyme for sequencing, various factors are considered. For example, large quantities of the enzyme should be easy to prepare; the enzyme should be stable upon storage for considerable time periods; the enzyme should accept all deoxy and dideoxy nucleotides and analogues as substrates with equal affinities and high fidelity; the polymerase activity should be highly processive over nucleotide extensions to 1 kb and beyond, even through regions of secondary structure within the template; the activity should remain high, even in suboptimal conditions; and the enzyme should be inexpensive (A. T. Bankier, “Dideoxy sequencing reactions using Klenow fragment DNA polymerase 1,” in H. and A. Griffin (eds.), Methods in Molecular Biology: DNA Sequencing Protocols, Humana Press, Totowa, N.J., 1993, pp. 83–90). Furthermore, the enzyme should be able to function at elevated temperatures (e.g., greater than about 70° C.), so that non-specific priming reactions are minimized. However, there are no native enzymes which fully meet all of these criteria. Thus, mutant forms of enzymes have been produced in order to address some of these needs.
For example, mutant forms of thermostable DNA polymerases that exhibit reduced or absent 5′ to 3′ exonuclease activity have been generated. The Stoffel fragment of Taq DNA polymerase lacks 5′ to 3′ exonuclease activity due to genetic manipulations that resulted in the production of a truncated protein lacking the N-terminal 289 amino acids (e.g., Lawyer et al., J. Biol. Chem., 264:6427–6437, 1989; and Lawyer et al., PCR Meth. Appi., 2:275–287, 1993). Analogous mutant polymerases have been generated from various polymerases, including Tma, Tsps17, TZ05, Tth and Taf. While the generation of thermostable polymerases lacking 5′ to 3′ exonuclease activity provides improved enzymes for certain applications, some of these mutant polymerases still have undesirable characteristics, including the presence of 3′ to 5′ exonuclease activity.
The 3′ to 5′ exonuclease activity is commonly referred to as proof-reading activity, it removes bases that are mismatched at the 3′ end of a primer in a primer-template duplex. While the presence of 3′ to 5′ exonuclease activity may be advantageous, as it leads to an increase in the fidelity of replication of nucleic acid strands, it also has some undesirable characteristics. The 3′ to 5′ exonuclease activity found in thermostable DNA polymerases such as Tma (including mutant forms of Tma that lack 5′ to 3′ exonuclease activity) also degrades single-stranded DNA such as primers used in PCR, single-stranded templates and single-stranded PCR products. The integrity of the 3′ end of an oligonucleotide primer used in a primer extension process (e.g., PCR, Sanger sequencing methods, etc.) is critical, as it is from this terminus that extension of the nascent strand begins. Degradation of the 3′ end of a primer results in loss of specificity in the priming reaction (i.e., the shorter the primer, the more likely that non-specific priming will occur).
Degradation of an oligonucleotide primer by a 3′ to 5′ exonuclease can be prevented by use of nucleotides modified at their 3′ terminus. For example, use of dideoxynucleotides or deoxynucleotides having a phosphorothiolate linkage between nucleotides at the 3′ terminus of an oligonucleotide can prevent degradation by 3′ to 5′ exonucleases. However, the need to use modified nucleotides to prevent degradation of oligonucleotides by a 3′ to 5′ exonuclease increases the time and cost required to prepare oligonucleotide primers.
A few examples of thermostable polymerases lacking both 5′ to 3′ exonuclease and 3′ to 5′ exonuclease are known. As discussed above, the Stoffel fragment of Taq DNA polymerase lacks the 5′ to 3′ exonuclease activity due to genetic manipulation and no 3′ to 5′ activity is present, as Taq polymerase is naturally lacking in 3′ to 5′ exonuclease activity. Likewise, Tth polymerase naturally lacks 3′ to 5′ exonuclease activity and deletion nucleotide sequence encoding N-terminal amino acids can be used to remove 5′ to 3′ exonuclease activity.
Despite development of recombinant enzymes such as Stoffel fragment, there remains a need for other thermostable polymerases having improved characteristics for various applications. For example, some thermostable polymerases possess reverse transcriptase activity and they find use in reverse transcription methods since elevated temperatures help the enzyme to proceed through regions of the RNA which at lower temperatures would possess secondary structure. However, reverse transcription by thermostable DNA polymerases is often dependent on manganese. Unfortunately, the presence of manganese ions can cause higher rates of infidelity and damage to polynucleotides. Accordingly, what is needed in the art are improved thermostable DNA polymerases with enhanced properties, such as reverse transcriptase activity in the presence of magnesium.